Modeling the impact of xenointoxication in dogs to halt Trypanosoma cruzi transmission.

BACKGROUND: Chagas disease, a vector-borne parasitic disease caused by Trypanosoma cruzi, affects millions in the Americas. Dogs are important reservoirs of the parasite. Under laboratory conditions, canine treatment with the systemic insecticide fluralaner demonstrated efficacy in killing Triatoma infestans and T. brasiliensis, T. cruzi vectors, when they feed on dogs. This form of pest control is called xenointoxication. However, T. cruzi can also be transmitted orally when mammals ingest infected bugs, so there is potential for dogs to become infected upon consuming infected bugs killed by the treatment. Xenointoxication thereby has two contrasting effects on dogs: decreasing the number of insects feeding on the dogs but increasing opportunities for exposure to T. cruzi via oral transmission to dogs ingesting infected insects. OBJECTIVE: Examine the potential for increased infection rates of T. cruzi in dogs following xenointoxication. DESIGN/METHODS: We built a deterministic mathematical model, based on the Ross-MacDonald malaria model, to investigate the net effect of fluralaner treatment on the prevalence of T. cruzi infection in dogs in different epidemiologic scenarios. We drew upon published data on the change in percentage of bugs killed that fed on treated dogs over days post treatment. Parameters were adjusted to mimic three scenarios of T. cruzi transmission: high and low disease prevalence and domestic vectors, and low disease prevalence and sylvatic vectors. RESULTS: In regions with high endemic disease prevalence in dogs and domestic vectors, prevalence of infected dogs initially increases but subsequently declines before eventually rising back to the initial equilibrium following one fluralaner treatment. In regions of low prevalence and domestic or sylvatic vectors, however, treatment seems to be detrimental. In these regions our models suggest a potential for a rise in dog prevalence, due to oral transmission from dead infected bugs. CONCLUSION: Xenointoxication could be a beneficial and novel One Health intervention in regions with high prevalence of T. cruzi and domestic vectors. In regions with low prevalence and domestic or sylvatic vectors, there is potential harm. Field trials should be carefully designed to closely follow treated dogs and include early stopping rules if incidence among treated dogs exceeds that of controls.

Local age-dependent neuromodulation in Rhodnius prolixus antennae.

Kissing bugs do not respond to host cues when recently molted and only exhibit robust host-seeking several days after ecdysis. Behavioral plasticity has peripheral correlates in antennal gene expression changes through the week after ecdysis. The mechanisms regulating these peripheral changes are still unknown, but neuropeptide, G-protein coupled receptor, nuclear receptor, and takeout genes likely modulate peripheral sensory physiology. We evaluated their expression in antennal transcriptomes along the first week postecdysis of Rhodnius prolixus 5th instar larvae. Besides, we performed clustering and co-expression analyses to reveal relationships between neuromodulatory (NM) and sensory genes. Significant changes in transcript abundance were detected for 50 NM genes. We identified 73 sensory-related and NM genes that were assigned to nine clusters. According to their expression patterns, clusters were classified into four groups: two including genes up or downregulated immediately after ecdysis; and two with genes with expression altered at day 2. Several NM genes together with sensory genes belong to the first group, suggesting functional interactions. Co-expression network analysis revealed a set of genes that seem to connect with sensory system maturation. Significant expression changes in NM components were described in the antennae of R. prolixus after ecdysis, suggesting that a local NM system acts on antennal physiology. These changes may modify the sensitivity of kissing bugs to host cues during this maturation interval.

Transovarial transmission of a core virome in the Chagas disease vector Rhodnius prolixus.

Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus, a primary vector of Chagas disease in Central and South America. We show that the RpVs belong to the Iflaviridae, Permutotetraviridae and Solemoviridae and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the permutotetraviruses and sobemoviruses display shared pools of vsiRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines.

Bacteriolytic activity in saliva of the hematophagous Triatoma infestans (Reduviidae) and novel characterization and expression site of a third lysozyme.

Saliva of hematophagous insects contains many different compounds, mainly acting as anticoagulants. Investigating the bacteriolytic compounds of the saliva of the bloodsucking Triatoma infestans photometrically between pH 3 and pH 10 using unfed fifth instars and nymphs up to 15 days after feeding, we found bacteriolytic activity against lyophilized Micrococcus luteus was stronger at pH 4 and pH 6. After feeding, the activity level at pH 4 was unchanged, but at pH 6 more than doubled between 3 and 7 days after feeding. In zymographs of the saliva and after incubation at pH 4, bacteriolytic activity against Micrococcus luteus was present at eight lysis zones between 14.1 and 38.5 kDa, showing the strongest activity at 24.5 kDa. After incubation at pH 6, lysis zones only appeared at 15.3, 17, and 31.4 kDa. Comparing zymographs of the saliva of unfed and fed nymphs, bacteriolytic activity at 17 kDa increased after feeding. In total nine lysis bands appeared, also at >30 kDa, so far unreported in the saliva of triatomines. Reverse transcription polymerase chain reaction using oligonucleotides based on the previously described lysozyme gene of T. infestans, TiLys1, verified expression of genes encoding TiLys1 and TiLys2 in the salivary glands, but also of an undescribed third lysozyme, TiLys3, of which the cloned cDNA shares characteristics with other c-type lysozymes of insects. While TiLys1 was expressed in the tissue of all three salivary glands, transcripts of TiLys2 and of TiLys3 seem to be present only in the gland G1 and G3, respectively.

Shelter selection in the kissing bugs Triatoma infestans and Rhodnius prolixus: Intra- and inter-specific interactions and quality evaluation.

Triatomines display most of their activities during the night. Before sunrise, they search, select and occupy adequate shelters to stay during the photophase, avoiding exposure to diurnal predators. In this work, we first explored the interactions between individuals of the same or different species in the shelter selection process of Triatoma infestans (Klug 1834) and Rhodnius prolixus (Stål 1859). When two groups of insects (either of different nutritional status, nymphal instar or species) were released together over an experimental arena containing two identical shelters, all nymphs were distributed randomly, suggesting the absence of intra- and inters-pecific interactions. Secondly, we analysed their preferences for particular features of shelters by releasing one group of insects (either T. infestans or R. prolixus) over an arena containing two different refuges. Nymphs exhibited preferences for darker shelters with a vertical orientation of its substrate and elevated from the ground, highlighting the importance of such features in a shelter selection context. We conclude that these species disregard the presence of other individuals but evaluate certain characteristics of the shelters to choose them. This information may contribute to understanding the colonization/recolonization dynamic processes of these Chagas disease vectors.

Molecular and cytogenetic evidence for sibling species in the Chagas disease vector Triatoma maculata.

Triatoma maculata (Hemiptera, Reduviidae, Triatominae) occurs across dry-to-semiarid ecoregions of northern South America, where it transmits Trypanosoma cruzi, causative agent of Chagas disease. Using 207 field-caught specimens from throughout the species' range, mitochondrial(mt) DNA sequence data, and cytogenetics, we investigated inter-population genetic diversity and the phylogenetic affinities of T. maculata. Mitochondrial DNA sequence analyses (cytb and nd4) disclosed a monophyletic T. maculata clade encompassing three distinct geographic groups: Roraima formation (Guiana shield), Orinoco basin, and Magdalena basin (trans-Andean). Between-group cytb distances (11.0-12.8%) were larger than the ~7.5% expected for sister Triatoma species; the most recent common ancestor of these T. maculata groups may date back to the late Miocene. C-heterochromatin distribution and the sex-chromosome location of 45S ribosomal DNA clusters both distinguished Roraima bugs from Orinoco and Magdalena specimens. Cytb genealogies reinforced that T. maculata is not sister to Triatoma pseudomaculata and probably represents an early (middle-late Miocene) offshoot of the 'South American Triatomini lineage'. In sum, we report extensive genetic diversity and deep phylogeographic structuring in T. maculata, suggesting that it may consist of a complex of at least three sibling taxa. These findings have implications for the systematics, population biology, and perhaps medical relevance of T. maculata sensu lato.

Lethal and repellent effect of amitraz, eugenol and thymol against Triatoma infestans, the main vector of Trypanosoma cruzi in the southern of America.

The lethal and repellent effect of the synthetic insecticide amitraz and the botanical insecticides eugenol and thymol separately and together in binary mixtures was tested against late-stage nymphs of a susceptible strain of Triatoma infestans, the main vector of Trypanosoma cruzi, the etiological agent of Chagas disease, in the Southern Cone of America. For the lethality study, the LD50 was determined for each insecticide alone and in binary mixture by topical application. The combination index (CI) was established to quantify interactions occurring between the insecticides. The repellent effect was tested using the area preference technique. The lethal effect of amitraz was 11 and 34 times more potent than that of thymol and eugenol, respectively. Only the combination of eugenol and amitraz at high concentrations showed a synergistic effect (CI: 0.3). The repellent activity of monoterpenes after 30 min of exposure was significant at 780 and 78 µg/cm2 for eugenol and thymol, respectively. The residual repellent effect of eugenol lasted for one week at the concentrations of 1170 and 1560 µg/cm2 , whereas thymol managed to retain its repellent effect for two weeks at concentrations of 1560 and 3900 µg/cm2 .

Extended stage duration and diminished fecundity in deltamethrin-resistant Triatoma infestans (Klug, 1834) of the Argentinean Chaco.

Pyrethroid-resistance is an emergent trait in populations of various insect species. For Triatoma infestans (Klug, 1834) (Heteroptera: Reduviidae), the major vector of Chagas disease in the southern part of South America, hotspot areas of pyrethroid-resistance have recently been found in the Chaco Province of Argentina. Resistant condition can reduce fitness of individuals in the absence of insecticide exposure, that is, fitness costs. We evaluated the existence of developmental and/or reproductive costs in T. infestans collected from two areas of pyrethroid-resistance in Chaco Province, Argentina. Three toxicological groups were defined from field-collected insects: susceptible (survival <20%), moderately resistant (survival between 20% and 80%) and highly resistant (survival >80%). Cohorts of the three toxicological groups were followed-up to study life cycle and reproductive parameters. Additionally, we parameterized matrix population growth models. First and IV nymphal stages of the resistant groups exhibited a longer stage duration than susceptible ones. The reproductive days and hatching success showed significant lower values revealing reproductive costs for the resistant groups. Matrix analysis showed lower population growth rates for the resistant groups. Our results support developmental and reproductive costs for pyrethroid-resistant individuals. This trait could be interpreted as lower population recovery ability for pyrethroid-resistant individuals compared to susceptible insects after alternative vector control actions.

Wild mammals involved in the transmission of Trypanosoma cruzi and food sources of Triatoma sherlocki in an endemic region of northeastern Brazil.

The present study, carried out in the municipality of Gentio do Ouro, Bahia, Brazil aimed to evaluate which wild mammals may be involved in the transmission of T. cruzi and which are the blood sources for triatomines collected in the study area. PCR analysis of 31 wild mammals captured revealed T. cruzi infection in 6.4% (2/31): one specimen of the opossum Didelphis albiventris (1/3) and one of the rodent Kerodon rupestris (1/5); despite being more frequent in the area, no specimen of the rodent Thrichomys sp. (0/23) was infected. A total of 169 triatomines were captured. The conclusive detection of food sources was possible only for Triatoma sherlocki Papa et al., 2002 (n = 56), with evidence for: K. rupestris (35.7%), Gallus (17.9%), D. albiventris (14.3%), Homo sapiens (14.3%), Tropidurus hispidus (7.1%), Leopardus geoffroyi (5.3%), Conepatus semistriatus (1.8%), Thrichomys inermis (1.8%) and Rattus norvegicus (1.8%). Triatomines of the species T. sherlocki showed food eclecticism, including feeding on humans, with some of them being captured at dwellings. These facts make this triatomine a potential link for the transmission of T. cruzi between wild and anthropic environments, highlighting a latent risk of the reemergence of Chagas disease outbreaks.

Influence of blood meal source on the biological parameters of Triatoma pallidipennis (Heteroptera: Reduviidae) from Mexico.

Chagas disease is one of the most important vector-borne diseases in Latin America, including Mexico. Triatoma pallidipennis (Stål) (Hemiptera: Reduviidae) is a Mexican triatomine vector commonly associated with different hosts. The influence of six blood meals (rabbits, rats, mice, dogs, cats and chickens) on six biological parameters of the biology of T. pallidipennis was evaluated. A significant difference was found in the period of egg-to-adult development between the five mammalian feeds (mean 195 days) and the chicken feed (221 days). The probability of survival was significantly lower in the chicken cohort (0.285). The total number of blood meals to moult from the first instar to the adult stage was the highest in the chicken cohort (10-15). This cohort had the significantly highest rate of females at the end cycle. The mean number of eggs laid per female and the egg eclosion rate were similar among the six food sources. Most results seemed to be influenced by the higher nutritional quality of the mammalian blood compared to the bird's blood and the increased energy expenditure required for the digestion of bird blood. These results clearly show that T. pallidipennis, unlike other triatomine species, has a high reproductive capacity when feeding on different hosts.

An increased number of individuals of a potential host facilitates non-photic synchronisation in the haematophagous insect Triatoma infestans.

BACKGROUND: Triatoma infestans (Kissing bug) is the main vector of the parasite causative of Chagas disease in Latin-America. This species shows clear activity rhythms easily synchronised to day-night cycles (photic cycle). The haematophagous nature of these insects lead us to think that they may temporally adapt to the particular activity rhythms of potential hosts (non-photic cycle). Our previous data showed that kissing bugs were weakly affected by the activity-inactivity rhythm of a single host. OBJETIVE: To determine if by increasing the number of individuals of a potential host, T. infestans could increase the likelihood of synchronisation. METHODS: Individual activity rhythms of experimental insects, maintained in constant darkness in light-tight cabinets, localised in a room with 24 rodents, were continuously monitored. Another insect group that served as control was maintained in the same conditions but in a room without rodents. FINDINGS: Most of the experimental insects synchronised, expressing a 24 h period coincident with the activity-inactivity rhythms of the rodents, while the controls free ran with a period significantly longer than 24 h. CONCLUSION: Analogous to what happens with high vs low light intensity in photic synchronisers, a high number of rodents, in contrast to the previous one-rodent experiment, increased the potency of this non-photic zeitgeber.

Molecular identification of Trypanosoma cruzi in domestic animals in municipalities of the State of Rio Grande do Norte, Brazil.

Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector.

The antennal transcriptome of Triatoma infestans reveals substantial expression changes triggered by a blood meal.

BACKGROUND: Triatoma infestans is the main vector of Chagas disease in the Americas, currently transmitting it in Argentina, Paraguay, and Bolivia. Many T. infestans populations present insecticide resistance, reducing the efficiency of control campaigns. Alternative vector control methods are needed, and molecular targets mediating fundamental physiological processes can be a promising option to manipulate kissing bug behavior. Therefore, it is necessary to characterize the main sensory targets, as well as to determine whether they are modulated by physiological factors. In order to identify gene candidates potentially mediating host cue detection, the antennal transcripts of T. infestans fifth instar larvae were sequenced and assembled. Besides, we evaluated whether a blood meal had an effect on transcriptional profiles, as responsiveness to host-emitted sensory cues depends on bug starvation. RESULTS: The sensory-related gene families of T. infestans were annotated (127 odorant receptors, 38 ionotropic receptors, 11 gustatory receptors, 41 odorant binding proteins, and 25 chemosensory proteins, among others) and compared to those of several other hemipterans, including four triatomine species. Several triatomine-specific lineages representing sensory adaptations developed through the evolution of these blood-feeding heteropterans were identified. As well, we report here various conserved sensory gene orthogroups shared by heteropterans. The absence of the thermosensor pyrexia, of pickpocket receptor subfamilies IV and VII, together with clearly expanded takeout repertoires, are revealed features of the molecular bases of heteropteran antennal physiology. Finally, out of 2,122 genes whose antennal expression was significantly altered by the ingestion of a blood meal, a set of 41 T. infestans sensory-related genes (9 up-regulated; 32 down-regulated) was detected. CONCLUSIONS: We propose that the set of genes presenting nutritionally-triggered modulation on their expression represent candidates to mediate triatomine host-seeking behavior. Besides, the triatomine-specific gene lineages found represent molecular adaptations to their risky natural history that involves stealing blood from an enormously diverse set of vertebrates. Heteropteran gene orthogroups identified may represent unknown features of the sensory specificities of this largest group of hemipteroids. Our work is the first molecular characterization of the peripheral modulation of sensory processes in a non-dipteran vector of human disease.

Lizards as Silent Hosts of Trypanosoma cruzi.

We assessed 4 lizard species in Chile for Trypanosoma cruzi, the causative agent of Chagas disease, and 1 species for its ability to transmit the protozoan to uninfected kissing bugs. All lizard species were infected, and the tested species was capable of transmitting the protozoan, highlighting their role as T. cruzi reservoirs.

Distribution patterns of Triatominae (Hemiptera: Reduviidae) in the Americas: an analysis based on networks and endemicity.

Triatominae, commonly known as kissing bugs, are a group of approximately 150 species of hematophagous reduviids, some of which are vectors of Trypanosoma cruzi, the etiological agent of the Chagas disease. Distributional patterns of triatomines have been studied based on macroecological and historical biogeographic approaches, but the definition of distributional patterns and areas of endemism are yet to be defined based on objective criteria. We used two methods to identify biogeographic units in the Triatominae: the endemicity analysis based on an optimality criterion (NDM/VNDM software) and a network approach aimed to simplify and highlight the underlying structure in species distributions (Infomap Bioregions). Information on species distributions was obtained from a data paper, comprising 21 815 records for 135 triatomine species occurring in the Americas. The resulting areas of each method were clustered using a meta consensus criterion based on dissimilarities and interpreted as recurrent areas. The NDM areas show a nested structure, presenting greater restrictions to the inclusion of species in a given area, requiring broad sympatry. In contrast, bioregions emphasize spatial patterns with better-delimited areas and species occurrences do not need to be highly congruent. When areas were clustered based on their species composition two clear patterns arose from both methods: (i) areas within the southern Amazon and southeast South America, especially in the Chacoan subregion, formed a cluster, and (ii) areas north of the Amazon, Pacific, Mesoamerica, Mexican transition zone and Nearctic formed another cluster. Moreover, within each of these two clusters, there was a latitudinal gradient of the areas in the Americas showing spatial similarity between the areas found in both methods. Results of both methods show well-bound areas separating the triatomine fauna in the Brazilian subregions, resulting in the recognition of areas corresponding to the biomes Chaco, Pampa, Cerrado, and Caatinga, and, to a lesser extent, the Atlantic Forest.

Sublethal effects of a pyrethroid insecticide on cuticle thickness, wing size and shape in the main vector Triatoma infestans.

Exposure to sublethal doses of insecticide may affect biological traits in triatomines. We investigated the effects of toxicological phenotype (pyrethroid resistance status) and exposure to sublethal doses of deltamethrin on two traits of Triatoma infestans Klug (Heteroptera: Reduviidae) using a phenotypic plasticity experimental design. First-instar nymphs from 14 and 10 full-sib families from pyrethroid-susceptible and pyrethroid-resistant populations, respectively, were used. For the susceptible population, we treated first instars topically with acetone (control) or deltamethrin (treatment) once. For the resistant population, instars were treated once, twice and three times as first, third or fifth-instar nymphs, respectively. We measured cuticle thickness, wing size and wing shape of 484 emerging adults, and tested for treatment effects using mixed ANOVA and MANOVA models. Toxicological phenotype, exposure to deltamethrin and full-sib family exerted significant effects on cuticle thickness, wing size and wing shape. Adult triatomines previously treated with deltamethrin developed significantly thicker cuticles than control triatomines only in the resistant population and significantly bigger wings in both populations. Mean cuticle thickness and wing size increased with increasing exposures to deltamethrin. Exposure to sublethal doses of deltamethrin generated morphological modifications that may affect insect survival and flight dispersal, and hence may have evolutionary and epidemiological consequences.

Control of pyrethroid-resistant populations of Triatoma infestans, the main vector of Trypanosoma cruzi, by treating dogs with fluralaner in the Argentine Chaco.

We assessed whether fluralaner administered to outbred healthy dogs reduced or supressed site infestation and abundance of pyrethroid-resistant populations of Triatoma infestans Klug (Heteroptera: Reduviidae). We conducted a placebo-controlled before-and-after efficacy trial in 28 infested sites in Castelli (Argentine Chaco) over 10 months. All 72 dogs initially present received either an oral dose of fluralaner (treated group) or placebo (control group) at month 0 posttreatment (MPT). Preliminary results justified treating all 38 control-house dogs with fluralaner 1 month later, and 71 of 78 existing dogs at 7 MPT. Site-level infestation and triatomine abundance were evaluated using timed manual searches with a dislodging aerosol. In the fluralaner-treated group, infestation dropped significantly from 100% at baseline to 19% over 6-10 MPT whereas mean abundance fell highly significantly from 5.5 to 0.8-0.9 triatomines per unit effort. In the placebo group, site infestation and mean abundance remained stable between 0 and 1 MPT, and strongly declined after fluralaner administration from 13.0-14.7 - triatomines at 0-1 MPT to 4.0-4.2 over 6-10 MPT. Only one of 81 noninfested sites before fluralaner treatment became infested subsequently. Fluralaner significantly reduced the site-level infestation and abundance of pyrethroid-resistant T. infestans and should be tested more widely in Phase III efficacy trials.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae).

BACKGROUND: Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS: Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES: To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS: We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS: Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

"Natural infections" with Trypanosoma cruzi via the skin of mice: size of mouthparts of vectors and numbers of invading parasites.

Investigating parameters influencing natural infections with Trypanosoma cruzi via the skin, the diameters of mouthparts of different stages of triatomines vectors were measured to determine the size of the channel accessible for T. cruzi during cutaneous infection. The mean diameters of the skin-penetrating mandibles of first to fifth instar nymphs of the vector Triatoma infestans increased from 18 to 65 µm. The mean diameter in fourth instar nymphs of Dipetalogaster maxima was 86 µm. Different numbers of isolated vector-derived metacyclic trypomastigotes (10-10,000) were injected intradermally into mice. Prepatent periods, parasitemia and mortality rates were compared with those of mice obtaining 10,000 metacyclic trypomastigotes that are usually present in the first drop of faeces onto the feeding wounds of fifth and fourth instar nymphs of T. infestans and D. maxima, respectively. After injection of 50-10,000 T. cruzi, in all 42 mice the infection developed. An injection of 10 parasites induced an infection in 8 out of 15 mice. With increasing doses of parasites, prepatent periods tended to decrease. The level of parasitemia was higher after injection of the lowest dose. Except for one mouse all infected mice died. After placement of 10,000 metacyclic trypomastigotes onto the feeding wound of fifth or fourth instar nymphs of T. infestans and D. maxima, respectively, the infection rates of the groups, prepatent periods and the levels of parasitemia of T. cruzi in mice indicated that about 10-1,000 metacyclic trypomastigotes entered the skin via this route. For the first time, the present data emphasise the risk of an infection by infectious excreta of triatomines deposited near the feeding wound and the low number of invading parasites.

Genetic diversity in peridomiciliary populations of Triatoma mexicana (Hemiptera: Reduviidae: Triatominae) in central Mexico.

Triatoma mexicana is an important vector of Trypanosoma cruzi-the etiological agent of Chagas disease. This triatomine species occurs in central Mexico, but little is known about its genetic variability. Using Cyt-b gene as a genetic marker, in this study, we determined the population genetic structure of T. mexicana collected from the States of Hidalgo, Guanajuato, and Queretaro where populations are largely peridomiciliary. A Bayesian approach was performed for the design of phylogenies, median-joining networks, and clustering among populations of T. mexicana. Our results show that the Hidalgo population was the most distinct, with the highest genetic and haplotypic variation (Hd = 0.963, π = 0.06129, and ɵ = 0.05469). Moderate gene flow (Nm) was determined among populations of Hidalgo and Queretaro. Populations from the three states showed differentiation (FST) values ranging from 0.22 to 0.3, suggesting an important genetic differentiation. The phylogenetic analysis showed the presence of five well-defined groups, as well as the haplotype network, where 24 haplotypes were observed forming five haplogroups with high mutational steps among them: 68 (Hgo-W2), 26 (Qto), 59 (Hgo-M), 44 (Hgo-W1), and 46 (Gto). Genetic isolation was apparently inferred in the Guanajuato population; however, the Mantel test did not show correlation between genetic (FST) and geographic (km) distances (p = 0.05). The STRUCTURE analyses showed seven genetic clusters and it was observed that a single cluster predominates in each sampled location. However, genetic admixture was detected in four localities. Our results show evidence that there are multiple species within the collected sampling area.